Current combined antiretroviral therapies (cART) suppress viral levels in the blood but do not eradicate reservoirs of cells harboring integrated copies of HIV proviral genomes. These cells persist in part because the provirus maintains a latent state that evades the immune response and viral cytopathic effect. Approaches to clear reservoirs by reactivating latent cells have provided evidence that latency can be reversed in vivo, however reversal of latency alone has not been sufficient to reduce latent reservoirs. Efforts are now in place to couple latency reactivation with strategies to eradicate the infected cells ? such as by design and activation of more efficacious anti-HIV cytotoxic T lymphocytes (CTLs). Another key player is Nef, an accessory protein encoded by HIV, which is a primary focus of our proposed research. Because Nef inhibits the activity of anti- HIV CTLs, a potent inhibitor of this protein would help achieve HIV eradication. One of the main functions of Nef is the down-modulation of major histocompatibility complex class I encoded proteins (MHC-I), masking infection from the host immune system and allowing HIV infected cells to persist. Combination therapy with latency antagonists plus Nef inhibitors could act synergistically to clear HIV reservoirs. To date, no Nef inhibitor has achieved potent restoration of MHC-I in the presence of Nef. We developed a high-throughput assay to identify inhibitors of Nef-mediated MHC-I downregulation, and a screen of natural product extracts (NPEs) yielded 10 hits with Nef inhibitory activity. We identified a number of related compounds, as the active component in several of these extracts. The pure natural products potently restore surface expression of MHC- I in the presence of Nef without inhibiting its other activities. We tested a number of structurally related compounds within this natural product family and identified two that possess pM to nM potencies in human primary cells. Based on this strong preliminary data, we believe that further enhancing the Nef inhibitory activity of these molecules through analog development will yield a safe anti-Nef drug. Therefore, we plan to (A) optimize these inhibitors by further separating and characterizing the anti-Nef effect from off-target activities to identify a lead drug candidate for development and (B) determine the mechanism by which the inhibitor disrupts Nef-mediated MHC-I downmodulation so that optimization can be conducted more intelligently. These goals will be achieved through the following specific aims: (1) Conduct lead compound structural optimization to improve pharmaceutical properties. (2) Perform a detailed functional analysis of all promising analogs to identify ideal lead compounds and (3) Determine the mechanism by which the natural product-derived inhibitor disrupts Nef-mediated MHC-I downmodulation including target identification and biochemical studies. From this work, we expect to generate a new class of compounds that are potent Nef inhibitors with high pharmaceutical potential. The addition of Nef inhibitory compounds to current cART cocktails is expected to enhance immune clearance of viral reservoirs, leading to the long-elusive HIV cure.